We have developed a transdominant genetic methodology for isolating peptide, protein or RNA factors (collectively called peturbagens) that modulate biochemical targets within a cell to alter its phenotype. Because it is genetic approach, it can be applied in both the classical forward mode (from phenotype to genotype) to reverse mode (from gene to function). The main goal of the proposed Phase I research is to validate human glutamine: fructose-6-phosphate amidotransferase (hsGFA1), the rate- limiting enzyme that catalyzes the first reaction in the hexosamine pathway, as a pharmacologic target for insulin resistance. This will be achieved by the following steps: (1) Isolating peptides that bindhsGFA1 using a modified yeast two hybrid assay. (2) Determine which peptides inhibit hsGFA1 activity by examining the conditional lethality of gfa1-/pGaI-hsGFA1+ yeast, that are viable despite to loss of the endogenous gfa1 gene due to rescue by galactose- regulated expression of hsGFA1. Inhibitors of hsGFA1 in these yeast cells will be cytotoxic. (3) Expressing the hsGFA1 peptide inhibitors in insulin-responsive cultured HepG2 hepatic cells and3T3-L1 adipocytes and ascertaining effects on GFA activity and the hexosamine pathway, as well as cellular glucose transport. The successful completion of Phase I will enable downstream studies of gsGFA1 inhibitors in mouse models of type 2 diabetes and obesity. PROPOSED COMMERCIAL APPLICATIONS: Perturbagen technology offers a novel way to probe and validate the function of a protein within a cell or animal. Proof that inhibition of hsGFA1 is capable of reversing insulin resistance in mammalian cells, or mouse models of diabetes and obesity, would open the flood gates for the search of chemical antagonists of the enzyme. If so, the peptide binders themselves may be useful in a high-throughput screen involving the displacement of the peptide binder from hsGFA1 by a small molecule.